Listeria monocytogenes is a pathogenic gram-positive bacterium. Causing Listeriosis it is one of the most virulent food borne pathogens.
The detection of pathogens in samples like food samples or clinical samples like blood, tissue or feces is becoming increasingly important. However, in order to clearly identify and optionally to quantify the cells comprised in a sample reliable methods for their detection and quantification have to be provided.
Since the first implementation of polymerase chain reaction (PCR) this molecular biological tool has been developed to a method providing many opportunities for improved detection of micro-organisms.
An important prerequisite for PCR based methods to become internationally recognized standard is the internal amplification control (IAC). An IAC is a non-target DNA sequence present in the same sample tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative result is not definite as it can mean that there was no target sequence present but also that the amplification reaction was inhibited by various factors. Therefore the European Standardization Committee in collaboration with the International Standard Organisation (ISO) proposed a general guideline for diagnostic PCR that requires the presence of an IAC (ISO/DIS 22174).
The IAC should be competitive using the same primer binding sites as the target PCR and clearly distinguishable from the target DNA amplicons by means of length and fluorescence wavelength of the used probe dye in real-time PCR. In summary the characteristics of the control should be as close to the characteristics of the target nevertheless ensuring negligible interference on target detection.
The implementation of an IAC to PCR assays ensures the reliability of negative results; however, this is only true for the enzymatic reaction facilitating target amplification in PCR and for the quantitative and confirmative detection reaction based on the use of fluorescent probes in real-time PCR. However, preliminary methodical steps such as sample preparation and DNA-isolation/purification from the sample matrix are not covered by this kind of control and if at all, checked by external controls. This does not allow for control of single samples and thus negative results imply the possibility of false verification of the pathogen status of the investigated samples, e.g. food.
Therefore an internal sample process control (ISPC) is necessary which should cover all methodical steps which are necessary for reliable quantitative detection of pathogens with conventional or real-time PCR.
For detection of food and waterborne RNA viruses the use of Feline Calcivirus and the bacteriophage MS2 as internal controls has been reported (Mattison et al. (2009) Int. J. Food Microbiol. 132, 73-77; Di et al. (2010) J. Virol. Methods 165, 57-63; Dreier et al. (2005) J. Clin. Microbiol. 43, 4551-4557).
Murphy et al. (2007, Int. J. Food Microbiol. 120, 110-119) discloses a bacterial internal sample process control for the detection of Listeria monocytogenes and Salmonella enterica. This control is based on a recombinant E. coli strain including fragments of the green fluorescence gene (gfp) and the iroB gene of S. enterica. The control was not used for quantitative purposes and the underlying gram negative E. coli strain will exhibit different characteristics during sample preparation compared to L. monocytogenes. 
Consequently, there is a need for new reliable methods allowing the detection and quantification of the pathogen Listeria monocytogenes by real-time PCR in contaminated samples.
Unexpectedly, it has been found that this problem can be solved by using an IAC+, Δ-prfA L. monocytogenes EGDe strain as internal process control covering the whole detection process.